Immunotherapy of B-cell malignancies using anti-CD22 antibodies

ABSTRACT

B-cell malignancies, such as the B-cell subtype of non-Hodgkin&#39;s lymphoma and chronic lymphocytic leukemia, are significant contributors to cancer mortality. The response of B-cell malignancies to various forms of treatment is mixed. Traditional methods of treating B-cell malignancies, including chemotherapy and radiotherapy, have limited utility due to toxic side effects. Immunotherapy with anti-CD20 antibodies have also provided limited success. The use of antibodies that bind with the CD22 or CD19 antigen, however, provides an effective means to treat B-cell malignancies such as indolent and aggressive forms of B-cell lymphomas, and acute and chronic forms of lymphatic leukemias. Moreover, immunotherapy with anti-CD22 and/or anti-CD19 antibodies requires comparatively low doses of antibody protein, and can be used effectively in multimodal therapies.

This application is a continuation of application Ser. No. 09/965,796,filed Oct. 1, 2001, which is a continuation of application Ser. No.09/307,816, filed May 10, 1999, now U.S. Pat. No. 6,306,393, whichapplication is a continuation-in-part of application Ser. No.09/038,955, filed Mar. 12, 1998, now U.S. Pat. No. 6,183,744, whichapplication claims the benefit of Provisional Application No.60/041,506, filed Mar. 24, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to immunotherapeutic methods for treatingB-cell malignancies. In particular, this invention is directed tomethods for treating B-cell malignancies by administering comparativelylow doses of antibody that binds to the CD22 antigen or antibody thatbinds to the CD19 antigen. The present invention also is directed tomultimodal therapeutic methods in which anti-CD22 or anti-CD19administration is supplemented with chemotherapy, or by administrationof therapeutic proteins, such as immunoconjugates and antibody fusionproteins.

2. Background

B-Cell lymphomas, such as the B-cell subtype of non-Hodgkin's lymphoma,are significant contributors to cancer mortality. The response of B-cellmalignancies to various forms of treatment is mixed. For example, incases in which adequate clinical staging of non-Hodgkin's lymphoma ispossible, field radiation therapy can provide satisfactory treatment.Still, about one-half of the patients die from the disease. Devesa etal., J. Nat'l Cancer Inst. 79:701 (1987).

The majority of chronic lymphocytic leukemias are of B-cell lineage.Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990). This type ofB-cell malignancy is the most common leukemia in the Western world.Goodman et al., Leukemia and Lymphoma 22:1 (1996). The natural historyof chronic lymphocytic leukemia falls into several phases. In the earlyphase, chronic lymphocytic leukemia is an indolent disease,characterized by the accumulation of small maturefunctionally-incompetent malignant B-cells having a lengthened lifespan.

Eventually, the doubling time of the malignant B-cells decreases andpatients become increasingly symptomatic. While treatment can providesymptomatic relief, the overall survival of the patients is onlyminimally affected. The late stages of chronic lymphocytic leukemia arecharacterized by significant anemia and/or thrombocytopenia. At thispoint, the median survival is less than two years. Foon et al., AnnalsInt. Medicine 113:525 (1990). Due to the very low rate of cellularproliferation, chronic lymphocytic leukemia is resistant to treatment.

Traditional methods of treating B-cell malignancies, includingchemotherapy and radiotherapy, have limited utility due to toxic sideeffects. The use of monoclonal antibodies to direct radionuclides,toxins, or other therapeutic agents offers the possibility that suchagents can be delivered selectively to tumor sites, thus limitingtoxicity to normal tissues.

Antibodies against the CD20 antigen have been investigated for thetherapy of B-cell lymphomas. For example, a chimeric anti-CD20 antibody,designated as “IDEC-C2B8,” has activity against B-cell lymphomas whenprovided as unconjugated antibodies at repeated injections of dosesexceeding 500 mg per injection. Maloney et al., Blood 84:2457 (1994);Longo, Curr. Opin. Oncol. 8:353 (1996). About 50 percent ofnon-Hodgkin's patients, having the low-grade indolent form, treated withthis regimen showed responses. Therapeutic responses have also beenobtained using ¹³¹I-labeled B1 anti-CD-20 murine monoclonal antibodywhen provided as repeated doses exceeding 600 mg per injection. Kaminskiet al., N. Engl. J. Med. 329:459 (1993); Press et al., N. Engl. J. Med.329:1219 (1993); Press et al., Lancet 346:336 (1995). However, theseantibodies, whether provided as unconjugated forms or radiolabeledforms, have not shown objective responses in patients with the moreprevalent and lethal form of B-cell lymphoma, the intermediate oraggressive type.

A need exists to develop an immunotherapy for B-cell malignancies thatallows repeated administration of comparatively low doses of anantibody, and that is not limited by the necessity of adding a toxicagent for achieving a therapeutic response of significant duration

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for treating B-cell malignancies using comparatively low doses ofanti-CD22 and/or anti-CD 19 antibodies.

It is a further object of this invention to provide multimodal methodsfor treatment of B-cell malignancies in which low doses of anti-CD22and/or anti-CD19 antibodies are supplemented with the administration ofa therapeutic protein, such as an immunoconjugate or antibody fusionprotein, or by a chemotherapeutic regimen.

These and other objects are achieved, in accordance with one embodimentof the present invention, by the provision of a method of treating aB-cell malignancy, comprising the step of administering to a subjecthaving a B-cell malignancy an anti-CD22 antibody and a pharmaceuticallyacceptable carrier.

DETAILED DESCRIPTION 1. Overview

As discussed above, anti-CD20 antibodies, whether unconjugated orlabeled with a therapeutic radionuclide, have failed to provideobjective responses in patients with intermediate or aggressive forms ofB-cell lymphoma. Surprisingly, clinical studies with patients havingnon-Hodgkin's lymphoma (both indolent and aggressive forms) or acutelymphatic leukemia have demonstrated that relatively low doses (i.e.,20-100 mg protein per dose) of unconjugated murine or humanizedanti-CD22 antibody, designated as either “EPB-2” or “LL2,” can inducepartial or complete remissions lasting up to 24 months. This, despitethe fact that such patients are often in relapse after multiple coursesof aggressive chemotherapy, and even after bone marrow grafting. Thepositive results with unconjugated anti-CD22 antibody are particularlysurprising in advanced patients with the aggressive (intermediate) formof non-Hodgkin's lymphoma and in chronic and acute lymphatic leukemia,since unconjugated or radiolabeled anti-CD20 antibodies have failed toshow such effects, particularly at low protein doses. Moreover, thepositive results with anti-CD22 antibodies are unexpected in view of thestatement by Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990),that chronic lymphocytic leukemias of the B-cell type do not generallyexpress CD22.

2. Definitions

In the description that follows, and in the documents incorporated byreference herein, a number of terms are used extensively. The followingdefinitions are provided to facilitate understanding of the invention.

A structural gene is a DNA sequence that is transcribed into messengerRNA (mRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned antibody gene is aDNA fragment that has been separated from the genomic DNA of a mammaliancell. Another example of an isolated DNA molecule is achemically-synthesized DNA molecule that is not integrated in thegenomic DNA of an organism.

An enhancer is a DNA regulatory element that can increase the efficiencyof transcription, regardless of the distance or orientation of theenhancer relative to the start site of transcription.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-CD22 monoclonal antibody fragment bindswith an epitope of CD22.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“sFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A chimeric antibody is a recombinant protein that contains the variabledomains and complementary determining regions derived from a rodentantibody, while the remainder of the antibody molecule is derived from ahuman antibody.

Humanized antibodies are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a therapeutic agent is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A naked antibody is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term antibody component includes both an entireantibody and an antibody fragment.

An immunoconjugate is a conjugate of an antibody component with atherapeutic agent.

As used herein, the term antibody fusion protein refers to a recombinantmolecule that comprises one or more antibody components and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators (“antibody-immunomodulatorfusion protein”) and toxins (“antibody-toxin fusion protein”). Thefusion protein may comprise a single antibody component, a multivalentcombination of different antibody components or multiple copies of thesame antibody component.

3. Production of Anti-CD22 and Anti-CD19 Monoclonal Antibodies,Humanized Antibodies, Primate Antibodies and Human Antibodies

Rodent monoclonal antibodies to CD22 or CD19 can be obtained by methodsknown to those skilled in the art. See generally, for example, Kohlerand Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [“Coligan”]. Briefly, monoclonal antibodies can be obtained byinjecting mice with a composition comprising CD22 or CD19, verifying thepresence of antibody production by removing a serum sample, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce anti-CD22 or anti-CD19 antibodies, culturing theclones that produce antibodies to the antigen, and isolating theantibodies from the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Suitable amounts of the well-characterized CD22 or CD19 antigen forproduction of antibodies can be obtained using standard techniques. Asan example, CD22 can be immunoprecipitated from B-lymphocyte proteinusing the deposited antibodies described by Tedder et al., U.S. Pat. No.5,484,892 (1996).

Alternatively, CD22 protein or CD19 protein can be obtained fromtransfected cultured cells that overproduce CD22 or CD19. Expressionvectors that comprise DNA molecules encoding CD22 or CD19 proteins canbe constructed using published CD22 and CD 19 nucleotide sequences. See,for example, Wilson et al., J. Exp. Med. 173:137 (1991); Wilson et al.,J. Immunol. 150:5013 (1993). As an illustration, DNA molecules encodingCD22 or CD19 can be obtained by synthesizing DNA molecules usingmutually priming long oligonucleotides. See, for example, Ausubel etal., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to8.2.13 (1990) [“Ausubel”]. Also, see Wosnick et al., Gene 60:115 (1987);and Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rdEdition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995). Establishedtechniques using the polymerase chain reaction provide the ability tosynthesize genes as large as 1.8 kilobases in length. Adang et al.,Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCR Methods andApplications 2:266 (1993); Dillon et al., “Use of the Polymerase ChainReaction for the Rapid Construction of Synthetic Genes,” in METHODS INMOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS ANDAPPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc. 1993).

In a variation of this approach, anti-CD22 or anti-CD19 monoclonalantibody can be obtained by fusing myeloma cells with spleen cells frommice immunized with a murine pre-B cell line stably transfected withCD22 cDNA or CD19 cDNA. See Tedder et al., U.S. Pat. No. 5,484,892(1996).

One example of a suitable murine anti-CD22 monoclonal antibody is theLL2 (formerly EPB-2) monoclonal antibody, which was produced againsthuman Raji cells derived from a Burkitt lymphoma. Pawlak-Byczkowska etal., Cancer Res. 49:4568 (1989). This monoclonal antibody has anIgG_(2α) isotype, and the antibody is rapidly internalized into lymphomacells. Shih et al, Int. J. Cancer 56:538 (1994). Immunostaining and invivo radioimmunodetection studies have demonstrated the excellentsensitivity of LL2 in detecting B-cell lymphomas. Pawlak-Byczkowska etal., Cancer Res. 49:4568 (1989); Murthy et al., Eur. J. Nucl. Med.19:394 (1992). Moreover, ^(99m)Tc-labeled LL2-Fab′ fragments have beenshown to be useful in following upstaging or B-cell lymphomas, while¹³¹I-labeled intact LL2 and labeled LL2 F(ab′)₂ fragments have been usedto target lymphoma sites and to induce therapeutic responses. Murthy etal., Eur. J. Nucl. Med. 19:394 (1992); Mills et al., Proc. Am. Assoc.Cancer Res. 34:479 (1993) [Abstract 2857]; Baum et al., Cancer 73(Suppl. 3):896 (1994); Goldenberg et al., J. Clin. Oncol. 9:548 (1991).Furthermore, Fab′ LL2 fragments conjugated with a derivative ofPseudomonas exotoxin has been shown to induce complete remissions formeasurable human lymphoma xenografts growing in nude mice. Kreitman etal., Cancer Res. 53:819 (1993).

In an additional embodiment, an antibody of the present invention is achimeric antibody in which the variable regions of a human antibody havebeen replaced by the variable regions of a rodent anti-CD22 or anti-CD19antibody. The advantages of chimeric antibodies include decreasedimmunogenicity and increased in vivo stability.

Techniques for constructing chimeric antibodies are well-known to thoseof skill in the art. As an example, Leung et al., Hybridoma 13:469(1994), describe how they produced an LL2 chimera by combining DNAsequences encoding the V_(κ) and V_(H) domains of LL2 monoclonalantibody with respective human κ and IgG₁ constant region domains. Thispublication also provides the nucleotide sequences of the LL2 light andheavy chain variable regions, V_(κ) and V_(H), respectively.

In another embodiment, an antibody of the present invention is asubhuman primate antibody. General techniques for raisingtherapeutically useful antibodies in baboons may be found, for example,in Goldenberg et al., international patent publication No. WO 91/11465(1991), and in Losman et al., Int. J. Cancer 46: 310 (1990).

In yet another embodiment, an antibody of the present invention is a“humanized” monoclonal antibody. That is, mouse complementaritydetermining regions are transferred from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, followed bythe replacement of some human residues in the framework regions of theirmurine counterparts. Humanized monoclonal antibodies in accordance withthis invention are suitable for use in therapeutic methods. Generaltechniques for cloning murine immunoglobulin variable domains aredescribed, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988),Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437(1992), and Singer et al., J. Immun. 150:2844 (1993). The publication ofLeung et al., Mol. Immunol. 32:1413 (1995), describes the constructionof humanized LL2 antibody.

In another embodiment, an antibody of the present invention is a humanmonoclonal antibody. Such antibodies are obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994).

4. Production of Antibody Fragments

The present invention contemplates the use of fragments of anti-CD22 andanti-CD19 antibodies or other therapeutically useful antibodies.Antibody fragments can be prepared by proteolytic hydrolysis of anantibody or by expression in E. coli of the DNA coding for the fragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and referencescontained therein. Also, see Nisonoff et al., Arch Biochem. Biophys.89:230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., inMETHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), andColigan at pages 2.8.1-2.8.10 and 2.10.-2.10.4. Other methods ofcleaving antibodies, such as separation of heavy chains to formmonovalent light-heavy chain fragments, further cleavage of fragments,or other enzymatic, chemical or genetic techniques may also be used, solong as the fragments bind to the antigen that is recognized by theintact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described in Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, for example,Sandhu, supra.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains which areconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains which are connectedby an oligonucleotide. The structural gene is inserted into anexpression vector which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2:97 (1991). Also seeBird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No.4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu,supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991);Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995);and Ward et al., “Genetic Manipulation and Expression of Antibodies,” inMONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al.,(eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

5. Preparation of Immunoconjugates

The present invention contemplates the use of “naked” anti-CD22 andanti-CD19 antibodies, as well as the use of immunoconjugates to effecttreatment of B-cell malignancies. Such immunoconjugates can be preparedby indirectly conjugating a therapeutic agent to an antibody component.General techniques are described in Shih et al., Int. J. Cancer41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); andShih et al., U.S. Pat. No. 5,057,313. The general method involvesreacting an antibody component having an oxidized carbohydrate portionwith a carrier polymer that has at least one free amine function andthat is loaded with a plurality of drug, toxin, chelator, boron addends,or other therapeutic agent. This reaction results in an initial Schiffbase (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer is preferably an aminodextran or polypeptide of atleast 50 amino acid residues, although other substantially equivalentpolymer carriers can also be used. Preferably, the final immunoconjugateis soluble in an aqueous solution, such as mammalian serum, for ease ofadministration and effective targeting for use in therapy. Thus,solubilizing functions on the carrier polymer will enhance the serumsolubility of the final immunoconjugate. In particular, an aminodextranwill be preferred.

The process for preparing an immunoconjugate with an aminodextrancarrier typically begins with a dextran polymer, advantageously adextran of average molecular weight of about 10,000-100,000. The dextranis reacted with an oxidizing agent to effect a controlled oxidation of aportion of its carbohydrate rings to generate aldehyde groups. Theoxidation is conveniently effected with glycolytic chemical reagentssuch as NaIO₄, according to conventional procedures.

The oxidized dextran is then reacted with a polyamine, preferably adiamine, and more preferably, a mono- or polyhydroxy diamine. Suitableamines include ethylene diamine, propylene diamine, or other likepolymethylene diamines, diethylene triamine or like polyamines,1,3-diamino-2-hydroxypropane, or other like hydroxylated diamines orpolyamines, and the like. An excess of the amine relative to thealdehyde groups of the dextran is used to insure substantially completeconversion of the aldehyde functions to Schiff base groups.

A reducing agent, such as NaBH₄, NaBH₃CN or the like, is used to effectreductive stabilization of the resultant Schiff base intermediate. Theresultant adduct can be purified by passage through a conventionalsizing column to remove cross-linked dextrans.

Other conventional methods of derivatizing a dextran to introduce aminefunctions can also be used, e.g., reaction with cyanogen bromide,followed by reaction with a diamine.

The aminodextran is then reacted with a derivative of the particulardrug, toxin, chelator, immunomodulator, boron addend, or othertherapeutic agent to be loaded, in an activated form, preferably, acarboxyl-activated derivative, prepared by conventional means, e.g.,using dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof,to form an intermediate adduct.

Alternatively, polypeptide toxins such as pokeweed antiviral protein orricin A-chain, and the like, can be coupled to aminodextran byglutaraldehyde condensation or by reaction of activated carboxyl groupson the protein with amines on the aminodextran.

Chelators for radiometals or magnetic resonance enhancers are well-knownin the art. Typical are derivatives of ethylenediaminetetraacetic acid(EDTA) and diethylenetriaminepentaacetic acid (DTPA). These chelatorstypically have groups on the side chain by which the chelator can beattached to a carrier. Such groups include, e.g., benzylisothiocyanate,by which the DTPA or EDTA can be coupled to the amine group of acarrier. Alternatively, carboxyl groups or amine groups on a chelatorcan be coupled to a carrier by activation or prior derivatization andthen coupling, all by well-known means.

Boron addends, such as carboranes, can be attached to antibodycomponents by conventional methods. For example, carboranes can beprepared with carboxyl functions on pendant side chains, as is wellknown in the art. Attachment of such carboranes to a carrier, e.g.,aminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier to produce anintermediate conjugate. Such intermediate conjugates are then attachedto antibody components to produce therapeutically usefulimmunoconjugates, as described below.

A polypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier must have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andimmunoconjugate.

Conjugation of the intermediate conjugate with the antibody component iseffected by oxidizing the carbohydrate portion of the antibody componentand reacting the resulting aldehyde (and ketone) carbonyls with aminegroups remaining on the carrier after loading with a drug, toxin,chelator, immunomodulator, boron addend, or other therapeutic agent.Alternatively, an intermediate conjugate can be attached to an oxidizedantibody component via amine groups that have been introduced in theintermediate conjugate after loading with the therapeutic agent.Oxidation is conveniently effected either chemically, e.g., with NaIO₄or other glycolytic reagent, or enzymatically, e.g., with neuraminidaseand galactose oxidase. In the case of an aminodextran carrier, not allof the amines of the aminodextran are typically used for loading atherapeutic agent. The remaining amines of aminodextran condense withthe oxidized antibody component to form Schiff base adducts, which arethen reductively stabilized, normally with a borohydride reducing agent.

Analogous procedures are used to produce other immunoconjugatesaccording to the invention. Loaded polypeptide carriers preferably havefree lysine residues remaining for condensation with the oxidizedcarbohydrate portion of an antibody component. Carboxyls on thepolypeptide carrier can, if necessary, be converted to amines by, e.g.,activation with DCC and reaction with an excess of a diamine.

The final immunoconjugate is purified using conventional techniques,such as sizing chromatography on Sephacryl S-300.

Alternatively, immunoconjugates can be prepared by directly conjugatingan antibody component with a therapeutic agent. The general procedure isanalogous to the indirect method of conjugation except that atherapeutic agent is directly attached to an oxidized antibodycomponent.

It will be appreciated that other therapeutic agents can be substitutedfor the chelators described herein. Those of skill in the art will beable to devise conjugation schemes without undue experimentation.

As a further illustration, a therapeutic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. For example, the tetanus toxoid peptides can be constructedwith a single cysteine residue that is used to attach the peptide to anantibody component. As an alternative, such peptides can be attached tothe antibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J.Cancer 56:244 (1994). General techniques for such conjugation arewell-known in the art. See, for example, Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,“Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

As described above, carbohydrate moieties in the Fc region of anantibody can be used to conjugate a therapeutic agent. However, the Fcregion is absent if an antibody fragment is used as the antibodycomponent of the immunoconjugate. Nevertheless, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofan antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995).The engineered carbohydrate moiety is then used to attach a therapeuticagent.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalf-life of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a “divalent immunoconjugate” by attaching therapeutic agentsto a carbohydrate moiety and to a free sulfhydryl group. Such a freesulfhydryl group may be located in the hinge region of the antibodycomponent.

6. Preparation of Fusion Proteins

The present invention contemplates the use of fusion proteins comprisingone or more antibody moieties and an immunomodulator or toxin moiety.Useful antibody moieties include antibody components that bind withCD19, CD20, CD22, CD52 or CD74, and a fusion protein may comprise one,two, three, four or all five of these antibody types. Bivalent,trivalent, tetravalent and quintavalent constructs can be used inaccordance with the invention.

Methods of making antibody-immunomodulator fusion proteins are known tothose of skill in the art. For example, antibody fusion proteinscomprising an interleukin-2 moiety are described by Boleti et al., Ann.Oncol. 6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995),Becker et al., Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al.,Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res. 56:4998(1996). In addition, Yang et al., Hum. Antibodies Hybridomas 6:129(1995), describe a fusion protein that includes an F(ab′)₂ fragment anda tumor necrosis factor alpha moiety. Moreover, the therapeutic use ofan hLL2-IL-2 fusion protein is illustrated by Example 5 of the presentapplication.

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch conjugates are ricin, abrin, ribonuclease, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journalfor Clinicians 44:43 (1994). Other suitable toxins are known to those ofskill in the art.

7. Coupling of Antibodies, Immunoconjugates and Fusion Proteins to LipidEmulsions

Long-circulating sub-micron lipid emulsions, stabilized withpoly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE), can beused as drug carriers for the anti-CD22 and anti-CD19 antibodycomponents, immunoconjugates, and fusion proteins of the presentinvention. The emulsions are composed of two major parts: an oil core,e.g., triglyceride, stabilized by emulsifiers, e.g., phospholipids. Thepoor emulsifying properties of phospholipids can be enhanced by adding abiocompatible co-emulsifier such as polysorbate 80. In a preferredembodiment, the anti-CD22 and anti-CD19 antibody components,immunoconjugates and fusion proteins are conjugated to the surface ofthe lipid emulsion globules with a poly(ethylene glycol)-based,heterobifunctional coupling agent, poly(ethyleneglycol)-vinylsulfone-N-hydroxy-succinimidyl ester (NHS-PEG-VS).

The submicron lipid emulsion is prepared and characterized as described.Lundberg, J. Pharm. Sci., 83:72 (1993); Lundberg et al., Int. J. Pharm.,134:119 (1996). The basic composition of the lipid emulsion istriolein:DPPC:polysorbate 80, 2:1:0.4 (w/w). When indicated, PEG-DPPE isadded into the lipid mixture at an amount of 2-8 mol % calculated onDPPC.

The coupling procedure starts with the reaction of the NHS ester groupof NHS-PEG-VS with the amino group of distearoylphosphatidyl-ethanolamine (DSPE). Twenty-five μmol of NHS-PEG-VS arereacted with 23 μmol of DSPE and 50 μmol triethylamine in 1 ml ofchloroform for 6 hours at 40° C. to produce a poly(ethylene glycol)derivative of phosphatidyl-ethanolamine with a vinylsulfone group at thedistal terminus of the poly(ethylene glycol) chain (DSPE-PEG-VS). Forantibody conjugation, DSPE-PEG-VS is included in the lipid emulsion at 2mol % of DPPC. The components are dispersed into vials from stocksolutions at −20° C., the solvent is evaporated to dryness under reducedpressure. Phosphate-buffered saline (PBS) is added, the mixture isheated to 50° C., vortexed for 30 seconds and sonicated with a MSE probesonicator for 1 minute. Emulsions can be stored at 4° C., and preferablyare used for conjugation within 24 hours.

Coupling of anti-CD22 or anti-CD19 antibodies to emulsion globules isperformed via a reaction between the vinylsulfone group at the distalPEG terminus on the surface of the globules and free thiol groups on theantibody. Vinylsulfone is an attractive derivative for selectivecoupling to thiol groups. At approximately neutral pH, VS will couplewith a half life of 15-20 minutes to proteins containing thiol groups.The reactivity of VS is slightly less than that of maleimide, but the VSgroup is more stable in water and a stable linkage is produced fromreaction with thiol groups.

Before conjugation, the antibody is reduced by 50 mM 2-mercaptoethanolfor 10 minutes at 4.degree. C. in 0.2 M Tris buffer (pH 8.7). Thereduced antibody is separated from excess 2-mercaptoethanol with aSephadex G-25 spin column, equilibrated in 50 mM sodium acetate buffered0.9% saline (pH 5.3). The product is assayed for protein concentrationby measuring its absorbance at 280 nm (and assuming that a 1 mg/mlantibody solution of 1.4) or by quantitation of .sup.125I-labeledantibody. Thiol groups are determined with ALDRITHIOL™ (2,2′-dipyridyldisulfide) following the change in absorbance at 343 nm and with cysteinas a standard.

The coupling reaction is performed in HEPES-buffered saline (pH 7.4)overnight at ambient temperature under argon. Excess vinylsulfone groupsare quenched with 2 mM 2-mercaptoethanol for 30 minutes, excess2-mercaptoethanol and antibody are removed by gel chromatography on aSepharose CL-48 column. The immunoconjugates are collected near the voidvolume of the column, sterilized by passage through a 0.45 μm sterilefilter, and stored at 4° C.

Coupling efficiency is calculated using ¹²⁵I-labeled antibody. Recoveryof emulsions is estimated from measurements of [¹⁴C]DPPC in parallelexperiments. The conjugation of reduced LL2 to the VS group ofsurface-grafted DSPE-PEG-VS is very reproducible with a typicalefficiency of near 85%.

8. Therapeutic Use of Anti-CD22 and Anti-CD19 Antibodies in Simple andMultimodal Regimens

The present invention contemplates the use of naked anti-CD22 andanti-CD19 antibodies, or immunoconjugates or fusion proteins comprisinganti-CD22 or anti-CD19 antibodies, as the primary therapeuticcomposition for treatment of B-cell malignancies. Such a composition cancontain polyclonal anti-CD22 or anti-CD19 antibodies or monoclonalanti-CD22 or anti-CD19 antibodies.

In addition, a therapeutic composition of the present invention cancontain a mixture of monoclonal anti-CD22 antibodies directed todifferent, non-blocking CD22 epitopes, or a mixture of monoclonalanti-CD19 antibodies directed to different, non-blocking CD19 epitopes.Monoclonal antibody cross-inhibition studies have identified fiveepitopes on CD22, designated as epitopes A-E. See, for example,Schwartz-Albiez et al., “The Carbohydrate Moiety of the CD22 Antigen CanBe Modulated by Inhibitors of the Glycosylation Pathway,” in LEUKOCYTETYPING IV. WHITE CELL DIFFERENTIATION ANTIGENS, Knapp et al. (eds.), p.65 (Oxford University Press 1989). As an illustration, the LL2 antibodybinds with epitope B. Stein et al., Cancer Immunol. Immunother. 37:293(1993). Accordingly, the present invention contemplates therapeuticcompositions comprising a mixture of monoclonal anti-CD22 antibodiesthat bind at least two CD22 epitopes. For example, such a mixture cancontain monoclonal antibodies that bind with at least two CD22 epitopesselected from the group consisting of epitope A, epitope B, epitope C,epitope D and epitope E. Similarly, the present invention contemplatestherapeutic compositions comprising a mixture of monoclonal anti-CD 19antibodies that bind at least two CD 19 epitopes.

Methods for determining the binding specificity of an anti-CD22 antibodyare well-known to those of skill in the art. General methods areprovided, for example, by Mole, “Epitope Mapping,” in METHODS INMOLECULAR BIOLOGY, VOLUME 10: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.),pages 105-116 (The Humana Press, Inc. 1992). More specifically,competitive blocking assays to determine CD22 epitope specificity aredescribed by Stein et al., Cancer Immunol. Immunother. 37:293 (1993),and by Tedder et al., U.S. Pat. No. 5,484,892 (1996).

The Tedder patent also describes the production of CD22 mutants whichlack one or more immunoglobulin-like domains. These mutant proteins wereused to determine that immunoglobulin-like domains 1, 2, 3, and 4correspond with epitopes A, D, B, and C, respectively. Thus, CD22epitope specificity can also be identified by binding a test antibodywith a panel of CD22 proteins lacking particular immunoglobulin-likedomain.

Although naked anti-CD22 antibodies or anti-CD19 antibodies are theprimary therapeutic compositions for treatment of B-cell malignancies,the efficacy of such antibody therapy can be enhanced by supplementingnaked antibodies with immunoconjugates, fusion proteins, and other formsof supplemental therapy described herein. In such multimodal regimens,the supplemental therapeutic compositions can be administered before,concurrently or after administration of the naked anti-CD22 or anti-CD19antibodies.

The therapeutic compositions described herein are particularly usefulfor treatment of indolent forms of B-cell lymphomas, aggressive forms ofB-cell lymphomas, chronic lymphatic leukemias, and acute lymphaticleukemias. For example, anti-CD22 antibody components andimmunoconjugates can be used to treat both indolent and aggressive formsof non-Hodgkin's lymphoma.

A radiolabeled antibody, immunoconjugate or fusion protein may comprisean α-emitting radioisotope, a β-emitting radioisotope, a γ-emittingradioisotope, an Auger electron emitter, a neutron capturing agent thatemits α-particles or a radioisotope that decays by electron capture.Suitable radioisotopes include ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ⁶⁷Cu, ²¹¹At, ²¹³Bi, ²²⁴Ac, and the like.

As discussed above, a radioisotope can be attached to an antibodycomponent directly or indirectly, via a chelating agent. For example,⁶⁷Cu, considered one of the more promising radioisotopes forradioimmunotherapy due to its 61.5 hour half-life and abundant supply ofbeta particles and gamma rays, can be conjugated to an antibodycomponent using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,“Medical Applications of Radioisotopes,” in REMINGTON'S PHARMACEUTICALSCIENCES, 18th Edition, Gennaro et al. (eds.), pages 624-652 (MackPublishing Co. 1990). Alternatively, ⁹⁰Y, which emits an energetic betaparticle, can be coupled to an antibody component usingdiethylenetriaminepentaacetic acid (DTPA). Moreover, a method for thedirect radiolabeling of the antibody component with ¹³¹I is described byStein et al., Antibody Immunoconj. Radiopharm. 4: 703 (1991).

Alternatively, boron addends such as carboranes can be attached toantibody components, as discussed above.

Preferred immunoconjugates and fusion proteins include antibodycomponents and conjugates of an anti-CD22 or anti-CD19 antibodycomponent and an immunomodulator. As used herein, the term“immunomodulator” includes cytokines, stem cell growth factors,lymphotoxins, such as tumor necrosis factor (TNF), and hematopoieticfactors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3,IL-6, IL-10 and IL-12), colony stimulating factors (e.g.,granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β and -γ), the stem cell growth factor designated “S1factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, interferon-γ,TNF-α, and the like. Alternatively, subjects can receive naked anti-CD22or naked anti-CD19 antibodies and a separately administered cytokine,which can be administered before, concurrently or after administrationof the naked anti-CD22 or anti-CD19 antibodies. The cytokines enhancethe activity of ADCC/NK, the effector cells that effect kill of tumorcells by binding to the Fc domain of human IgG1 antibodies, a domainthat is present in hLL2.

Antibody-immunomodulator immunoconjugates and antibody-immunomodulatorfusion proteins provide a means to deliver an immunomodulator to atarget cell and are particularly useful against tumor cells. Thecytotoxic effects of immunomodulators are well known to those of skillin the art. See, for example, Klegerman et al., “Lymphokines andMonokines,” in BIOTECHNOLOGY AND PHARMACY, Pessuto et al. (eds.), pages53-70 (Chapman & Hall 1993). As an illustration, interferons can inhibitcell proliferation by inducing increased expression of class Ihistocompatibility antigens on the surface of various cells and thus,enhance the rate of destruction of cells by cytotoxic T lymphocytes.Furthermore, tumor necrosis factors, such as TNF-α, are believed toproduce cytotoxic effects by inducing DNA fragmentation.

Useful cancer chemotherapeutic drugs for the preparation ofimmunoconjugates and fusion proteins include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidineanalogs, purine analogs, antibiotics, epipodophyllotoxins, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985). Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

In addition, therapeutically useful immunoconjugates can be obtained byconjugating photoactive agents or dyes to an antibody composite.Fluorescent and other chromogens, or dyes, such as porphyrins sensitiveto visible light, have been used to detect and to treat lesions bydirecting the suitable light to the lesion. In therapy, this has beentermed photoradiation, phototherapy, or photodynamic therapy (Jori etal. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (LibreriaProgetto 1985); van den Bergh, Chem. Britain 22:430 (1986)). Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. Mew et al., J. Immunol. 130:1473 (1983); idem.,Cancer Res. 45:4380 (1985); Oseroff et al., Proc. Natl. Acad. Sci. USA83:8744 (1986); idem., Photochem. Photobiol. 46:83 (1987); Hasan et al.,Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med.9:422 (1989); Pelegrin et al., Cancer 67:2529 (1991). However, theseearlier studies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes. Multimodal therapies of thepresent invention further include immunotherapy with naked anti-CD22 andnaked anti-CD19 antibodies supplemented with administration of anti-CD19and anti-CD22 antibodies, respectively, as well as with theco-administration of anti-CD20, CD52 and/or CD74 antibodies in the formof naked antibodies or as immunoconjugates. Anti-CD 19 and anti-CD20antibodies are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., CancerImmunol. Immunother. 32:364 (1991); Kaminski et al., N. Engl. J. Med.329:459 (1993); Press et al., N. Engl. J. Med. 329:1219 (1993); Maloneyet al., Blood 84:2457 (1994); Press et al., Lancet 346:336 (1995);Longo, Curr. Opin. Oncol. 8:353 (1996).

In another form of multimodal therapy, subjects receive naked anti-CD22or naked anti-CD19 antibodies, and/or immunoconjugates or fusionproteins, in conjunction with standard cancer chemotherapy. For example,“CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and 150-200mg/m² carmustine) is a regimen used to treat non-Hodgkin's lymphoma.Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitablecombination chemotherapeutic regimens are well-known to those of skillin the art. See, for example, Freedman et al., “Non-Hodgkin'sLymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.(eds.), pages 2028-2068 (Lea & Febiger 1993). As an illustration, firstgeneration chemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine,procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate and brostatin-1. In a preferred multimodal therapy, bothchemotherapeutic drugs and cytokines are co-administered with anantibody, immunoconjugate or fusion protein according to the presentinvention. The cytokines, chemotherapeutic drugs and antibody,immunoconjugate or fusion protein can be administered in any order, ortogether.

In general, the dosage of administered anti-CD22 and anti-CD19antibodies, anti-CD22 and anti-CD19 antibody components,immunoconjugates, and fusion proteins will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Typically, it is desirable toprovide the recipient with a dosage of antibody component,immunoconjugate or fusion protein which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate.

Administration of antibody components, immunoconjugates or fusionproteins to a patient can be intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses.

Those of skill in the art are aware that intravenous injection providesa useful mode of administration due to the thoroughness of thecirculation in rapidly distributing antibodies. Intravenousadministration, however, is subject to limitation by a vascular barriercomprising endothelial cells of the vasculature and the subendothelialmatrix. Still, the vascular barrier is a more notable problem for theuptake of therapeutic antibodies by solid tumors. Lymphomas haverelatively high blood flow rates, contributing to effective antibodydelivery. Intralymphatic routes of administration, such as subcutaneousor intramuscular injection, or by catheterization of lymphatic vessels,also provide a useful means of treating lymphomas.

Preferably, naked anti-CD22 or anti-CD19 antibodies are administered atlow protein doses, such as 20 to 1500 milligrams protein per dose, givenonce, or repeatedly, parenterally. Alternatively, naked anti-CD22 oranti-CD19 antibodies are administered in doses of 20 to 1000 milligramsprotein per dose, or 20 to 500 milligrams protein per dose, or 20 to 100milligrams protein per dose.

As described above, the present invention also contemplates therapeuticmethods in which naked anti-CD22 or anti-CD 19 antibody components aresupplemented with immunoconjugate or fusion protein administration. Inone variation, naked anti-CD22 or anti-CD19 antibodies are administeredwith low-dose radiolabeled anti-CD22 or anti-CD19 antibodies orfragments. As a second alternative, naked anti-CD22 or anti-CD19antibodies are administered with low-dose radiolabeledanti-CD22-cytokine or anti-CD19-cytokine immunoconjugates. As a thirdalternative, naked anti-CD22 or anti-CD19 antibodies are administeredwith anti-CD22-cytokine or anti-CD19-cytokine immunoconjugates that arenot radiolabeled. With regard to “low doses” of ¹³¹I-labeledimmunoconjugates, a preferable dosage is in the range of 15 to 40 mCi,while the most preferable range is 20 to 30 mCi. In contrast, apreferred dosage of ⁹⁰Y-labeled immunoconjugates is in the range from 10to 30 mCi, while the most preferable range is 10 to 20 mCi. Preferredantibody components include antibodies and fragments derived from LL2antibodies, including murine LL2 monoclonal antibody, chimeric LL2antibody, and humanized LL2 antibody.

Immunoconjugates having a boron addend-loaded carrier for thermalneutron activation therapy will normally be effected in similar ways.However, it will be advantageous to wait until non-targetedimmunoconjugate clears before neutron irradiation is performed.Clearance can be accelerated using an antibody that binds to theimmunoconjugate. See U.S. Pat. No. 4,624,846 for a description of thisgeneral principle.

The anti-CD22 and anti-CD19 antibody components, immunoconjugates andfusion proteins alone, or conjugated to liposomes, can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic proteins are combined in a mixturewith a pharmaceutically acceptable carrier. A composition is said to bea “pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (1995).

For purposes of therapy, antibody components (or immunoconjugates/fusionproteins) and a pharmaceutically acceptable carrier are administered toa patient in a therapeutically effective amount. A combination of anantibody component, optionally with an immunoconjugate/fusion protein,and a pharmaceutically acceptable carrier is said to be administered ina “therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. In the present context, an agent is physiologicallysignificant if its presence results in the inhibition of the growth oftarget tumor cells.

Additional pharmaceutical methods may be employed to control theduration of action of an antibody component, immunoconjugate or fusionprotein in a therapeutic application. Control release preparations canbe prepared through the use of polymers to complex or adsorb theantibody component, immunoconjugate or fusion protein. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10:1446 (1992).The rate of release of an antibody component (or immunoconjugate) fromsuch a matrix depends upon the molecular weight of the protein, theamount of antibody component/immunoconjugate/fusion protein within thematrix, and the size of dispersed particles. Saltzman et al., Biophys.J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th ed. (1995).

The present invention also contemplates a method of treatment in whichimmunomodulators are administered to prevent, mitigate or reverseradiation-induced or drug-induced toxicity of normal cells, andespecially hematopoietic cells. Adjunct immunomodulator therapy allowsthe administration of higher doses of cytotoxic agents due to increasedtolerance of the recipient mammal. Moreover, adjunct immunomodulatortherapy can prevent, palliate, or reverse dose-limiting marrow toxicity.Examples of suitable immunomodulators for adjunct therapy include G-CSF,GM-CSF, thrombopoietin, IL-1, IL-3, IL-12, and the like. The method ofadjunct immunomodulator therapy is disclosed by Goldenberg, U.S. Pat.No. 5,120,525.

For example, recombinant IL-2 may be administered intravenously as abolus at 6×10⁵ IU/kg or as a continuous infusion at a dose of 18×10⁶IU/m²/d. Weiss et al., J. Clin. Oncol. 10:275 (1992). Alternatively,recombinant IL-2 may be administered subcutaneously at a dose of 12×10⁶IU. Vogelzang et al., J. Clin. Oncol. 11:1809 (1993). Moreover, INF-γmay be administered subcutaneously at a dose of 1.5×10⁶ U. Lienard etal., J. Clin. Oncol. 10:52 (1992). Furthermore, Nadeau et al., J.Pharmacol. Exp. Ther. 274:78 (1995), have shown that a singleintravenous dose of recombinant IL-12 (42.5 μg/kilogram) elevated IFN-γlevels in rhesus monkeys.

Suitable interleukin-2 (IL-2) formulations include PROLEUKIN® (IL-2aldesleukin) (Chiron Corp./Cetus Oncology Corp.; Emeryville, Calif.) andTECELEUKIN® (rIL-2, recombinant interleukin-2) (Hoffmann-La Roche, Inc.;Nutley, N.J.). ACTIMMUNE® (Interferon gamma-1B, IFNγ-1B) (Genentech,Inc.; South San Francisco, Calif.) is a suitable INF-γ preparation.

EXAMPLE 1 Treatment of a Patient with Indolent Lymphoma in Lymph Nodesand Bone Marrow

A patient presents with diffuse, large cell, malignant lymphoma, in lungand liver. The patient has a good, but short, response to CHOP. Sevenmonths later, the patient receives high dose chemotherapy along with abone marrow transplant. Ten months later, the patient relapses, withlung, liver and lymphoadenopathy, and is treated with four standarddoses of RITUXAN® (rituximab). The patient had a brief response to theRITUXAN®, which lasted less than 3 months. The patient then failed asecond treatment with RITUXAN®, and was characterized as havingprogressive lymphoma with lung, liver and lymphoadenopathy (Day 0).

The patient then began therapy with humanized LL2 monoclonal antibody.The patient was infused intravenously with 634 mg of humanized LL2antibody, and the treatment was repeated 6, 13, and 20 days followingthis initial treatment. Immediately following the last dose, the serumvalue of hLL2 was 389.7 μg/ml, and one month following the last dose theserum value of hLL2 was 186.5 μg/ml.

Five months after the final dose of hLL2, a computerized tomography scanof the patient showed no evidence of lymphoma, resolution ofsplenomegaly, and no liver abnormality, and subsequent histology withimmunoperoxidase staining of paraffin tissue sections for CD20 and CD3reveals no evidence of lymphoma in bone marrow. Normal B-cells in theblood prior to therapy with hLL2 were completely depleted from the blood2 months post-therapy, and there was minimal reappearance of normal Bcells five months post-therapy. The results are shown in the followingtables.

TABLE 1 B-cells and T-cells in marrow % marrow % marrow % marrow B-cellsT-cells HLA-Dr Day CD19 CD20 Kappa lambda CD3 (Ia) Flow cytometry 0 1215 20 3 7 20 Conventional histology 0 30 and 40% malignant lymphomacells in two aspirates 28 hLL2 therapy 34 hLL2 therapy 41 hLL2 therapy48 hLL2 therapy Flow cytometry 203 3 1 1 <1 32 2 Immunoperoxidasestaining of paraffin tissue sections for CD20 and CD3 203 5 95Conventional histology 203 Small lymphoid aggregates/hypocellularitywith myeloid hypoplasia

TABLE 2 B-cells and T-cells in blood % blood % blood % blood B-cellsT-cells HLA-Dr Day T4/T8 CD19 CD20 kappa lambda CD3 (Ia) Flow cytometry0 1.5 5 5 6 2 38 6 28 hLL2 therapy 34 hLL2 therapy 41 hLL2 therapy 48hLL2 therapy Flow cytometry 76 1.3 <1 <1 <1 <1 71 6 191 2.0 1 1 <1 <1 734

EXAMPLE 2 Treatment of a Patient with Aggressive, Diffuse, Large CellLymphoma in Lung and Liver

A patient presents with diffuse, large cell, malignant lymphoma, in lungand liver. The patient has a good, but short, response to CHOP. Sevenmonths later, the patient receives high dose chemotherapy along with abone marrow transplant. Ten months later, the patient relapses, withlung, liver and lymphoadenopathy, and is treated with four standarddoses of Rituxan. The patient had a brief response to the Rituxan, whichlasted less than 3 months. The patient then failed a second treatmentwith Rituxan, and was characterized as having progressive lymphoma withlung, liver and lymphoadenopathy (Day 0).

The patient then began therapy with humanized LL2 monoclonal antibody.The patient was infused intravenously with 556 mg of humanized LL2antibody, and the treatment was repeated 5, 12, and 19 days followingthis initial treatment. Immediately following the last dose, the serumvalue of hLL2 was 279.8 μg/ml, and one month following the last dose theserum value of hLL2 was 99.1 μg/ml.

Prior to treatment, a CT scan of the patient showed three lung lesions,3.96, 4.83 and 4.6 cm², respectively. One month after the final dose ofhLL2, a CT scan of the patient showed the lesions were reduced to 0,1.21 and 0.81 cm², respectively. Four and a half months after the finaldose of hLL2, a CT scan showed the three lesions were reduced to 0, 1and 0 cm², respectively.

Normal B-cells in the blood prior to therapy were markedly reduced,probably due to the RITUXAN® therapy. There was minimal reappearance ofnormal B cells one month post-therapy. The results are shown in thefollowing tables.

TABLE 3 B-cells and T-cells in marrow % marrow % marrow % marrow B-cellsT-cells HLA-Dr Day CD19 CD20 kappa lambda CD3 (Ia) Flow cytometry 0 8020 Conventional histology 28 negative for lymphoma 28 hLL2 therapy 33hLL2 therapy 40 hLL2 therapy 47 hLL2 therapy

TABLE 4 B-cells and T-cells in blood % blood % blood % blood B-cellsT-cells HLA-Dr Day T4/T8 CD19 CD20 kappa lambda CD3 (Ia) Flow cytometry0 0.5 <1 <1 <1 <1 57 4 28 hLL2 therapy 33 hLL2 therapy 40 hLL2 therapy47 hLL2 therapy Flow cytometry 48 0.3 <3 1 <1 <1 68 4 76 0.4 <1 <1 1 163 15

TABLE 5 Results of CT scans Lesion Location Day 19 Day 50 Day 182 lesionsize in cm² Left axillary  6.82 4.18 resolved Portacaval 20.16 5.04resolved Retrocaval  5.72 3.24 resolved Paraaortic  4.00 2.88 resolved

EXAMPLE 3 Treatment of a Patient with Relapsed Intermediate-GradeNon-Hodgkin's Lymphoma

A patient with intermediate grade non-Hodgkin's lymphoma has failedprior aggressive chemotherapy, consisting of CHOP×6, which led to acomplete remission for five months, another course of CHOP×6, resultingin progression, D-MOPP×2, resulting in stable disease for six months,and CVB with peripheral stem cell transplantation, which led to apartial remission for four months. The patient presents with recurrentlymphoma in the chest and in a neck lymph node, both measurable bycomputerized tomography and palpation, respectively.

The patient is infused with 50 mg of humanized LL2 monoclonal antibodyon days 2, 5, 9, 12 of two successive weeks with no adverse effectsnoted. Three weeks later, palpation of the neck node enlargement shows ameasurable decrease of about 60%, while a repeat computerized tomographyscan of the chest shows a marked, 70% reduction in tumor. Follow-upmeasurements made at ten weeks post therapy shows no evidence of thedisease in the neck or the chest. Since new disease is not detectedelsewhere, the patient is considered to be in complete remission.Follow-up studies every 10-12 weeks confirms a complete remission for atleast 16 months post therapy.

EXAMPLE 4 Treatment of a Patient with Diffuse Large Cell AggressiveLymphoma with CHOP and hLL2

A patient presents with diffuse large cell aggressive lymphoma, and isdiagnosed to have a poor prognosis, having bulky disease in the abdomen,numerous other sites of extranodal disease, and elevated serum lactatedehydrogenase (LDH). The patient is placed on CHOP, and after threecycles of therapy, a partial response is observed with resolution ofnumerous sites of extranodal disease outside the abdomen. However, thebulky disease in the abdomen continues to increase in volume, and theserum LDH remains elevated.

Upon initiation of the third cycle of CHOP, the patient is infused with50 mg of humanized LL2 monoclonal antibody on days 2.5, 9 and 12. Thistherapeutic regimen of hLL2 is repeated concomitantly with four morecycles of CHOP. During therapy, the serum LDH level falls to within thenormal range. One month after the third cycle of CHOP and hLL2, acomputerized tomography scan of the bulky tumor in the abdomen showsover a 90% shrinkage of the mass. Follow-up studies every 10-12 weeksconfirms a complete remission for over nine months post-therapy.

EXAMPLE 5 Treatment of a Patient with Relapsed, Aggressive Large CellLymphoma with hLL2 and hLL2-IL2

A patient with diffuse large cell aggressive lymphoma responds to firstline (CHOP) and second line (m-BACOD) chemotherapy, but fails third linechemotherapy (MACOP-B). After completion of third line chemotherapy, thepatient has diffuse disease in the bone marrow, massive splenomegaly,and numerous sites of enlarged lymph nodes that could be palpitated. Thepatient is then infused with 50 mg of humanized LL2 on days 2, 5, 9 and12. This regimen is repeated every other week for four weeks. The bonemarrow disease progressively responds to the hLL2 treatment, and thesize of the nodes also decreases. However, many nodes can still bepalpitated, and little decrease is observed in spleen size. Whiletherapy with hLL2 continues every two weeks, the patient also receives10 mg of hLL2-IL2 fusion protein. After the first treatment, there is aprofound decrease in the size of the spleen, and after the secondtreatment with hLL2/hLL2-IL2, the nodes are not palpable, and the spleenhas decreased further in size. No progression of the disease is observedfor over six months.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those in the art to which theinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference in its entirety.

1. A method of treating a B-cell malignancy, comprising (i)administering an anti-CD20 monoclonal antibody or fragment thereof; and(ii) administering an anti-CD22 antibody or fragment thereof; whereinsaid anti-CD20 antibody or fragment and said anti-CD22 antibody orfragment are administered separately or in combination, and in eitherorder and wherein the antibody fragment is a synthetic, geneticallyengineered or naturally occurring isolated antibody fragment selectedfrom the group consisting of F(ab′), F(ab)₂, Fab′, Fab, Fv, and sFv. 2.A method according to claim 1, wherein said anti-CD20 monoclonalantibody or fragment is non-radiolabeled and said anti-CD22 monoclonalantibody or fragment is radiolabeled.
 3. A method according to claim 2,comprising (i) administering a non-radiolabeled anti-CD20 intactmonoclonal antibody; and (ii) administering a radiolabeled anti-CD22antibody or fragment thereof; wherein said anti-CD20 intact monoclonalantibody and said radiolabeled anti-CD22 antibody or fragment areadministered separately or in combination, and in either order andwherein the CD22 antibody fragment is a synthetic, geneticallyengineered or naturally occurring isolated antibody fragment selectedfrom the group consisting of F(ab′), F(ab)₂, Fab′, Fab, Fv, and sFv. 4.A method according to claim 2, comprising (i) administering atherapeutically effective amount of non-radiolabeled anti-CD20monoclonal antibody; and (ii) administering a therapeutically effectiveamount of a radiolabeled anti-CD22 antibody or fragment thereof; whereinsaid anti-CD20 antibody and said radiolabeled anti-CD22 antibody orfragment are administered separately or in combination, and in eitherorder.
 5. A method according to claim 4, wherein said anti-CD20 antibodyis rituximab.
 6. The method of claim 4, which is used to treat a B-cellmalignancy selected from the group consisting of leukemia and lymphoma.7. The method of claim 4, which is used to treat a B-cell malignancyselected from the group consisting of indolent forms of B-celllymphomas, aggressive forms of B-cell lymphomas, chronic lymphaticleukemias, and acute lymphatic leukemias.
 8. The method of claim 6,wherein said B-cell lymphoma is a non-Hodgkin's lymphoma.
 9. The methodof claim 4, wherein the amount of said anti-CD20 antibody ranges from20-1500 mg/dose.
 10. The method of claim 4, wherein the amount of saidanti-CD20 antibody ranges from 20-500 mg/dose.
 11. The method of claim4, wherein the amount of said anti-CD20 antibody ranges from 20-100mg/dose.
 12. The method of claim 4, wherein said radiolabeled anti-CD22antibody or fragment is an yttrium-90 labeled anti-CD22 antibody orfragment.
 13. The method of claim 4, wherein said radiolabeled anti-CD22antibody fragment is a F(ab)₂, F(ab′)₂, Fab, Fab′ or Fv.
 14. The methodof claim 4, wherein said anti-CD22 antibody is a ⁹⁰Y labeled humanizedLL2 antibody.
 15. The method of claim 14, wherein the dosage of saidradiolabeled antibody ranges from 10 to 30 mCi.
 16. The method of claim4, further comprising administering a chemotherapeutic agent.
 17. Themethod of claim 4, further comprising administering a cytokine.
 18. Themethod of claim 4, wherein said anti-CD20 antibody is a humanizedantibody.
 19. The method of claim 4, wherein said anti-CD20 antibody isa human antibody.
 20. The method of claim 1, wherein said anti-CD20antibody or fragment is a humanized antibody or antibody fragment. 21.The method of claim 1, wherein said anti-CD20 antibody or fragment is ahuman antibody or antibody fragment.
 22. The method according to claim1, wherein the anti-CD20 antibody is administered before the anti-CD22antibody.
 23. The method according to claim 1, wherein the anti-CD20antibody is administered concurrently with the anti-CD22 antibody. 24.The method according to claim 1, wherein the anti-CD22 antibody isadministered before the anti-CD20 antibody.